Method for the commercial production of helminths antigens

ABSTRACT

An easily cultured worm is genetically modified such that its surface antigens on its modified form are immunologically identical to the surface antigens of a parasitic worm. Specifically, C. elegans is genetically modified to produce antigens corresponding to the antigens of D. immitis which causes heartworm infection. The antigens derived from the easily cultured worm are used to form a vaccine and/or diagnostic.

This is a continuation of co-pending application Ser. No. 399,718 filedon July 19, 1982, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to the development and commercial production ofhelminths (parasitic worm) antigens.

The use of antigens for diagnostics and vaccines to detect and preventinfectious diseases such as polio, smallpox, diphtheria, tetanus andhoof and mouth diseases has been clearly shown in both humans andanimals. Most antigens used as diagnostics and vaccines are derived fromcultured infectious organisms. The infectious organisms are grown invitro or in vivo in animals or tissue cultures. An example is polioantigens which are derived from viruses grown in vitro in monkey kidneycells. The antigens are used as a diagnostic and to vaccinate humans.

If a small amount of attenuated viruses is inoculated in a host, theinoculated viruses which carry specific antigens will induce antibodyformation in the host which in the future will recognize and destroyfuture invading polio viruses. The same polio virus antigens can also beused for the diagnosis of a polio infection. If a host is infected bypolio, some specific antibodies will be elicited in the body and thepresence of these antibodies will be indicative of an infection. Thedetection of antibodies can be assayed for their binding to polioantigens with the standard immunofluoroscent, radio-immune, andenzyme-linked, immunoelectrophoresis, hemagglutinin and immunodiffusionassays.

It is not possible, however, to commercially produce antigens for allinfectious diseases. One limitation of production has been the inabilityto cultivate or produce a large amount of infectious organisms in vivoor in vitro from which the antigens are derived This is especially truefor vaccines in which the infectious organisms have complicated lifecycles and/or have more than one host. Most parasitic worm diseases thatinfect dogs, cats, sheep, pigs, horses and humans fall into thiscategory

An example is the helminths disease of the heartworm which can infect awide variety of organisms from dogs, cats, seals to humans(infrequently). The parasite, however, generally resides in the dog asits host with the mosquito as its intermediate host. It would bedifficult to derive antigens necessary in the manufacture of vaccines ordiagnostics since the parasitic worm undergoes several stages of larvaldevelopment and only one larval stage contains the appropriate antigens.In the heartworm this particular stage, the infectious larvae, residesin the mosquito. Under these conditions, the capture of the intermediatehosts (mosquitos) and the dissection for the infectious larvae isnecessary to generate required antigens for the manufacture of aheartworm vaccine. Wong, M. M., Guest, M. F., and Laviopierre, M. J.(1974) Dirofilaria immitis; Fate and Immunogenicity of IrradiatedInfective Stage Larvae in Beagles Experimental Parasitology 35, 65-74.Similarly, antigens which are necessary to diagnose heartworm antibodiesfor infection must be derived from adult heartworms harbored in theheart of an infected dog. Desowitz, R. S. and Una, S. R. (1976), TheDetection of Antibodies in Human and Animal Filariasis byCounter-immunoelectrophoresis with Dirofiliaria immitis Antigen. Journalof Helminthology, 50, 53-57, Grieve, R. B., Mika-Johnson, M., Jacobson,R. H., and Raymond, C. H., (1981) Enzyme-Linked Immunosorbent Assay forMeasurement of Antibody Response to Dirofilaria immitis inExperimentally Infected Dogs. American Journal of Veterinary Research,42, 66-69. These sources and methods of generating heartworm antigensfor diagnostics (from the heart of a dog) and vaccines (from mosquitos)are neither commercially feasible nor may they be socially acceptable.

I have devised a method of producing helminths (parasitic worm) antigenswherein a related species of the infectious worm that can be easilycultured is genetically modified. The method involves identifying thesurface antigens of the difficult to culture parasitic worm and thencreating these same antigens in the easily cultured species.

The easily cultured species is genetically altered through mutationsuntil said species have some of the same immunological antigens of thedifficult to culture parasitic worm. Through these geneticmanipulations, the types and amounts of antigens of interest in theeasily cultured species are altered. Antigens derived from thegenetically altered species are then used for the commercial productionof antigens necessary for the manufacture of diagnostics and vaccines.

The principle of my invention is to alter the antigen genes of theeasily cultured species to produce preferably in situ antigens of thehard to culture parasitic species. The expression of these altered genesto produce these antigens can also occur in bacteria or yeast with thecloning of the antigen genes, and thus, provide a potentially moreefficient production of these same antigens.

In the preferred embodiments, the antigens produced using this inventionagainst the heartworm infection is by Dirofilaria immitis, and thesubstituted related species is the free-living hermaphroditic nematodeCaenorhabditis elegans.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Generation of antibodies against heartworm antigens of Dirofilariaimmitis

Antibodies must be first generated against parasitic antigens ofinterest. These antibodies which recognize and bind specifically to thehard to culture parasitic antigens can then be used as a probe toisolate mutants in the easily cultured species which are capable ofproducing the corresponding parasitic antigens. In the examples setforth below, I have generated antibodies against adult heartwormantigens and isolated mutants in C. elegans with the correspondingheartworm antigens. Although described in reference to C. elegans, otherspecies which are believed suitable are Panagrellas redivious, Turbatricacetic and C. briggsae.

Adult heartworms were isolated from a dog infected with heartworm. Theadult heartworms were excised from the heart and suspended in 0.15 Mphosphate buffered saline pH 7.4 (PBS). The heartworms were homogenizedand the homogenate was adjusted to a final concentration of 500micrograms of protein (wet weight) per ml of PBS. This homogenate wasthen mixed with equal volume of Freund's complete adjuvant. A 2 mlsample of this mix was used to immunize a rabbit for heartworm antigens.Before actual immunization, a preimmune serum was drawn from the rabbit.The rabbit was inoculated at days 25 and 35 after the first immunizationwith 2 ml of a mixture of equal volume of homogenate and Freund'sincomplete adjuvant. After a positive test bleed at 45 days, the serumwas collected and used to assay for antibody formation against heartwormantigens used for the immunization.

Assay of serum containing anti-heartworm antibodies.

The serum of the rabbit immunized with heartworm antigens was assayedfor antibody activities against heartworm antigens and C. elegansantigens. My preferred method is to use a immunofluorescent assay ofconjugated fluoroscein or rhodamine goat anti-rabbit IgG. Other methodssuch as enzyme-linked, radioimmune, hemagglutinin and immundiffusionassay can also be used. Sections of heartworm tissues from dogs(containing the antigens) were washed three times with PBS and thenincubated with the rabbit anti-heartworm antibodies at room temperaturefor 30 minutes, the tissues were then washed 3 times with PBS to removethe rabbit anti-heartworm antibodies. Following this, the tissues wereincubated with fluoroscein goat anti-rabbit IgG antibodies. Afterincubation, the free goat anti-rabbit IgG antibodies were removed bywashing the heartworm tissues 3 times with PBS. The presence of rabbitantibody activities binding to specific heartworm antigens was indicatedby the binding of the fluoroscent goat anti-rabbit IgG antibodies whichwere then observed in a fluoroscent microscope, red (rhodamine) or green(fluorescein) in color.

C. elegans whole animals and tissues were also used to assay theanti-heartworm antibodies generated by the immunized rabbit serum. Theresults of the fluoroscent assays for anti-heartworm activities aresummarized in Table I.

The control preimmune serum had no activity against either heartwormtisues or C. elegans tissues. In contrast, there was evidence ofantibody activities in the immunized rabbit serum against heartwormtissues but not C. elegans. Thus, the generation of antibodies againstheartworm antigens is only specific for heartworm but not C. elegans.This serum which appears specific was used as a probe to isolate mutantsof C. elegans which will, in accordance with my invention, carryantigens corresponding to heartworm antigens.

                  TABLE I                                                         ______________________________________                                        Antibody activities of serum from a rabbit                                    which has been immunized with heartworm antigens                              Immune Serum        Preimmune Serum                                                  Heartworm C. elegans Heartworm                                                                             C. elegans                                Titer  tissue    tissue     tissue  tissue                                    ______________________________________                                        1:5    +         -          -       -                                         1:20   +         -          -       -                                         1:40   +         -          -       -                                         1:80   +         -          -       -                                          1:160 +         -          -       -                                         ______________________________________                                         + = fluorescent activity                                                      - = no activity                                                          

                  TABLE II                                                        ______________________________________                                        Antibody binding activity of six independently                                isolated mutants of C. elegans with                                           1:50 titer of rabbit anti-heartworm serum                                               Immune Serum                                                                            Preimmune Serum                                           ______________________________________                                        Wild Type   -           -                                                     (C. elegans)                                                                  Heartworm   +           -                                                     Mutant Strain                                                                 VGI-1       +           -                                                     VGI-2       +           -                                                     VGI-3       +           -                                                     VGI-4       +           -                                                     VGI-5       +           -                                                     VGI-6       +           -                                                     ______________________________________                                    

                  TABLE III                                                       ______________________________________                                        Antibody binding activity of one mutant VGI-6                                 with rabbit anti-heartworm serum                                                                             Wild Type                                              VGI-6        Heartworm (C. elegans)                                   Titer   tissue       tissue    tissue                                         ______________________________________                                        1:5     +            +         -                                              1:20    +            +         -                                              1:40    +            +         -                                              1:80    +            +         -                                               1:160  +            +         -                                              ______________________________________                                    

Mutagenization of C. elegans to generate mutants which have alteredantigens that correspond to heartworm antigens

The free-living soil nematode C. elegans is preferred since it can bereadily cultured in large quantities and homozygous recessive mutationscan be generated. Brenner, S. (1974), The Genetics of Caenorhabditiselegans Genetics 77, 71-94. This is due to the hermaphroditic nature ofthe animal, that is, it is self-fertilizing. A mutation introduced inthe animal upon self-fertilization for two generations will produce ahomozygous mutation.

Young larvae of C. elegans at the first and second larval stage ofdevelopment were exposed to the mutagen, ethyl methyl sulfonate (anyother mutagens can also be used, such as methyl methane sulfonate,acridine orange, nitrosoguanidine, hydroxynitrosamine; Drake, J. W. andR. H. Baltz (1976), The Biochemistry of Mutagenesis, Annual Review ofBiochemistry 45, 11) and allowed to self-fertilize and lay eggs. The F₁generation of this mutagenesis was allowed to reproduce and the F₂generation was then screened for animals which would bind to antibodiesmade against specific antigens of the heartworm. Because wild type C.elegans do not bind to antibodies made against heartworm antigensgenerated in our rabbit heartworm antiserum (See Table I), a visualscreen was made for mutants that had antigens that bound to theheartworm antibodies using the above fluorescent assay. Mutants whichbound to the heartworm antibodies unlike the wild type (with nofluorescence) had a fluorescent color of red (rhodamine) or green(fluorescein). These mutant animals were removed and cloned.

Animals which breed true for this characteristic are considered to bemutants which have an altered gene that would produce an antigen(s)corresponding to those of the heartworm.

Using the above methods, I have demonstrated with my invention, in TableII, that mutants of C. elegans with altered antigens corresponding tothose of heartworm antigens can be generated. The antibody bindingactivities with anti-heartworm serum of six (6) independently isolatedmutants is compared with heartworm tissues and wild type tissues inTable II.

Table III represents a further characterization of one of therepresentative mutants among the six (6) in Table II in which thebinding of antibody activity behave very closely to heartworm tissue.

Furthermore, when one of these C. elegans mutants was immunized to arabbit, it was able to elicit an immunogenic response in which theantibodies produced in the serum had binding activities to a species ofheartworm antigens.

Thus, the mutants isolated with my invention not only have an antigenicresponse of binding to heartworm antibodies, but also a immunogenicresponse in producing antibodies against heartworm antigens.

Using the above described method, I also isolated a mutant of C. eleganswhich was altered in at least some of its surface antigens to beimmunologically identical to the antigens of another parasitic worm,Ancylostoma caninum, a hookworm which infests in the intestine of thedog.

Any worm species which are readily cultured and in which mutations canbe introduced to cause an alteration in its antigens to a correspondingdifficult to culture parasitic worm antigen can be used with my method.

Conceptually, using C. elegans and the heartworm, D. immitis as anexample, once the antigens of the desired parasite are generated withthe above method, they can be used to develop a range of diagnostics forthe presence of antibodies induced upon a parasitic worm infection. Suchan approach with heartworm antigens has been shown to be useful in thediagnosis of several types of helminths infections. Desowitz and Una,supra; Hedge and Ridley (1977). Immunofluorescent Reactions withMicrofilariae. 1 Diagnostic Evaluation; Transaction of the Royal Societyof Tropical Medicine and Hygiene 71, 304-307. In the case of theheartworm antigens which I have generated, I have found that one of themutants isolated in C. elegans, VGI-2, is useful in the diagnosis ofheartworm infection. When serum samples of dogs diagnosed as infectedand uninfected with heartworms were assayed with the VGI-2 mutant, theresults were remarkedly accurate in that 6 dogs diagnosed with thestandard filter assay as positively infected based on the presence ofmicrofilarie were also found to be positive in this mutant with theimmunofluorescent assay. In contrast, 5 samples of serum from clinicallydiagnosed uninfected dogs were also found negative in the mutant.Besides the use of immunofluoroscent assay to detect parasitic worminfection with mutant antigens, enzyme-linked, radioimmune,immunodiffision or hemagglutinin assay can also be applied. D. Stites(1976), Laboratory Methods for Detection of Antigens and Antibodies,Basic and Clinical Immunology, Lange Medical Publications, pp. 281-315.

Antigens generated with the described method can also be used for themanufacture of parasitic worm vaccines. In the example of heartworminfection, it is known that the antigens derived from infectious larvaecan be used as a vaccine for dogs against heartworm infection; Wong, M.M., Guest, M. F., and Lavoipierre, M. J. (1974) Dirofilaria immitis;Fate and Immunogenicity of Irradiated Infective Stage Larvae in BeaglesExperimental Parasitology 35, 65-79. As long as antibodies can bespecifically generated against these particular infectious larvalantigens and mutants of C. elegans with corresponding antigens areisolated, these mutants can be used to derive the immunogenic antigensnecessary for the production and manufacture of heartworm vaccine. Thevaccine generated can then be suspended for example in a buffered salineand then used for injection subcutaneously or intramuscularly in ananimal for providing immunity against heartworm infection. W. J. Herbert(1978), Laboratory Animal Techniques for Immunology in "Handbook ofExperimental Immunology", Vol. 1 Immuno Chemistry, 3rd Edition, Editor,D. M. Weir, Blackwell Scientific Publishers, Oxford, London.

The basic principle of my method is to alter the gene(s) of a closelyrelated worm species that is easy to culture to generate antigens of adifficult to culture parasitic worm. In the described embodiment, theconcept of the generation of antigens are in situ in the species of theeasily cultured worm. This concept can be extended further in theexpression of antigens in another organism such as bacteria, yeast orother microorganisms which may provide a more efficient and productiveyield of the desired antigens. The isolation of the antigen gene(s) andtheir insertion into bacteria with genetic vectors can be applied. Thegenes for example in C. elegans can be isolated by initially isolatingthe nucleic acid of the animal with the proteinase K-SDS methoddescribed by Emmons, S., Klass, M. R., and Hirsh, D. (1979); Analysis ofthe Constancy of DNA Sequences During Development and Evolution of theNematode Caenorhabditis elegans Proceeding of the National Academy ofSciences 76, 1333-1337.

The poly A (RNA) is separated by ethanol precipitation and by binding tooligo(dT) cellulose according to Aviv and Leder (1972), Purification ofBiologically Active Globin Messenger RNA by Chroatography onOligothymidylic Acid Cellulose, Proceedings of the National Academy ofScience 69, 1408-1412. The material binding to oligo(dT) celulose arethat used to purify poly A (RNA) which codes for the antigen geneproduct. Since the poly A (RNA) will only hybridize to the DNA whichcodes for the antigen gene, this poly A (RNA) can be used to selectrecombinant poly A (DNA) molecules of C. elegans coding for the antigengene of interest. These recombinant DNA molecules can then be insertedinto bacteria or yeast with various genetic vector such as plasmids orbacteriophage to express the antigen gene and product of interest usingthe basic techniques described by Davis, R. W., Botstein, D., and Roth,J. R. (1980). A manual for genetic engineering--Advanced BacterialGenetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).

There is a possibility that antigens produced by the natural parasitesmay be more potent or specific than those originating from C. elegansthrough genetic mutations. Thus, the cloning of the naturally hard toculture parasitic genes for their antigens in an microorganism may beadvantageous. If the cloning approach described above is used, it wouldbe extremely difficult if not impossible to accomplish. This is due tothe limitation of the amount of parasitic tissues available to isolatethe poly A (RNA) of interest. The poly A (RNA) is only present intissues which are expressing the antigen. In the case of poly A (RNA)corresponding to the antigens of the infectious larvae of the heartworm,the tissues must be derived from larval animals dissected from capturedmosquitos which harbor the parasites. The availability of infectiouslarval tissues necessary to isolate enough poly A (RNA) corresponding totheir antigens is limited therefore, making the task of poly A (RNA)isolation extremely difficult, if not impossible. However, if we use thepoly A (RNA) isolated from C. elegans mutant that correspond to theseantigens of interest, we can through hybridization of the heartworm DNAisolate the recombinant DNA genes corresonding to the heartworminfectious larvae antigens. The availability of heartworm DNA is notlimited since all tissues have DNA containing all genes and a largequantity of tissue can be acquired from adult heartworms.

Having described my invention, what I now claim is:
 1. A method for theproduction of parasitic helminth antigens which comprises:identifyingthe antigens of D. immitis; modifying genetically C. elegans untilmutants are formed which mutants are characterized by antigenspossessing immunological identity to the antigens of D. immitis;isolating at least one of the mutants so modified and cultivating atleast one of said mutants.
 2. The method of claim 1 which includes:isolating the antigens of said mutants.
 3. The method of claim 1 whichincludes: identifying the mutants which have the immunological identitywith the antigens of the D. immitis by:isolating those mutants whichbind to antibodies generated by a host immunized with D. immitisantigens.
 4. A method for the production of parasitic helminth antigenswhich comprises:identifying the antigens of A. canium; modifyinggenetically C. elegans until mutants are formed which mutants arecharacterized by antigens possessing immunological identity to theantigens of A. canium; isolating at least one of the mutants so modifiedand cultivating at least one of said mutants.
 5. The method of claim 4which includes:isolating the antigens of said mutants.
 6. The method ofclaim 4 which includes:identifying the mutants which have theimmunological identity with the antigens of the A. canium by: isolatingthose mutants which bind to antibodies generated by a host immunizedwith A. canium antigens.
 7. A method for the production of parasitichelminth antigens which comprises:identifying the antigens of D.immitis; modifying genetically C. briggsae until mutants are formedwhich mutants are characterized by antigens possessing immunologicalidentity to the antigens of D. immitis; isolating at least one of themutants so modified and cultivating at least one of said mutants.
 8. Themethod of claim 7 which includes:isolating the antigens of said mutants.9. The method of claim 7 which includes:identifying the mutants whichhave the immunological identity with the antigens of the D. immitis by:isolating those mutants which bind to antibodies generated by a hostimmunized with D. immitis antigens.